The mammalian intestinal mucosa undergoes a process of continual renewal characterized by active proliferation of stem cells localized near the base of the crypts; progression of these cells up the crypt-villus axis with cessation of proliferation; and subsequent differentiation into one of the four primary cell types (i.e., enterocytes, goblet cells, Paneth cells and enteroendocrine cells). An imbalance in this highly-regimented and orderly process within the crypts is associated with a number of common intestinal pathologies (e.g., colorectal cancer and inflammatory bowel disease [IBD]). Delineating the molecular factors regulating intestinal proliferation and differentiation is crucial to our understanding of not only normal gut development and adaptation, but also aberrant gut growth. With the support of this grant, we have shown the importance of the PI3K/Akt/mTOR and Wnt/?-catenin pathways in the regulation of intestinal cell proliferation and differentiation. In addition, we showed that enhanced ketogenesis by mTORC1 inhibition contributes to the regulation of intestinal cell differentiation and that tuberous sclerosis complex 2/mTORC1 signaling contributes to the maintenance of intestinal epithelial homeostasis. Moreover, we found that activation of Wnt/?-catenin signaling suppresses the expression of the ketogenic enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2), which inhibits glycolysis in intestinal cells. Recently, we have shown that deficiency of SIRT2, a member of the sirtuin family of proteins, results in the activation of Akt/mTORC1 and Wnt/?-catenin, increased intestinal cell proliferation and stemness, and increased expression of glycolytic hexokinases (HK) 1 and 2. Using single cell sequencing, we found that HK1 and HK2 were differentially expressed in various types of intestinal cells; knockdown of HK2, but not HK1, increased Paneth and goblet cell differentiation. Based on our recently published and preliminary findings, we have further refined our central hypothesis to state that the glycolytic enzyme HK2, acting downstream of SIRT2, significantly contributes to the maintenance of normal intestinal homeostasis. To examine our central hypothesis, we have planned experiments with the following Specific Aims: 1) to further delineate the cellular mechanisms for SIRT2-mediated alterations of glycolysis and oxidative phosphorylation in intestinal cells; 2) to better define the role of HK2 in mediating the effects of SIRT2 in the intestine; and, 3) to determine the impact of alterations of SIRT2/HK2 on intestinal homeostasis in vivo. The studies in the current proposal represent direct and novel extensions of our previous findings, and are designed to define, in a systematic fashion, the molecular mechanisms and signaling events regulating the processes of intestinal proliferation, differentiation, and adaptation utilizing a number of innovative techniques including murine and human intestinal organoids, novel transgenic models, single-cell sequencing, and stable isotope resolved metabolomics (SIRM).